3 Prepare insert DNA Amplify by PCR Gel purify Digest Remove nucleotides Ligate Prepare vector DNA Digest Dephosphorylate Gel purify Verify clones Screen for expression Digest Sequence Transform BL21 (DE3) Grow several transformants in 2 5 ml cultures Induce with IPTG (when A 600 is ) for 6 8 h Harvest cells, lyse and analyze by SDS-PAGE Verify expression clone Figure 1 Outline of the protocol. Clone into second MCS of Duet? Large-scale expression Grow started culture overnight Innoculate 1 l with 1 5 ml of overnight culture Induce with IPTG (when A 600 is ) for 2 14 h Harvest cells, lyse and analyze by SDS-PAGE Affinity purification 11 If using both multiple cloning sites of the Duet vectors, repeat Steps 1 11 using a verified bacterial clone. (Optional) Small-scale test expression of the cloned target can be attempted on a 2 5-ml culture in LB and analyzed by SDS-PAGE to ensure the clone expresses and to determine solubility (Steps 12 22). 12 Use 1 µl (10 40 ng) of each plasmid to transform BL21 (DE3) competent cells. When transforming multiple plasmids, it is essential to allow the cells to recover for at least 1 h before plating on the LB agar containing the appropriate antibiotic (as determined by the chosen vector; Table 1). 13 Plate cells on LB agar containing the appropriate antibiotics (depending on the chosen vectors) and incubate at 37 C for h. In the case of multiple plasmids, reduce the concentration of antibiotics used by half of the values in Table 1. TROUBLESHOOTING 14 Pick several transformants and set up a 2 5 ml culture of each in LB containing appropriate antibiotics. Transformation of each clone into BL21 (DE3) Screening clones for expression 15 Grow the cultures to an A 600 of with vigorous shaking at C. 16 Remove 10 µl of culture for analysis by SDS-PAGE (preinduced sample) and another 1 ml for the preparation of a glycerol stock (add glycerol to a final concentration of 15%) and store at 70 C. Cold Spring Harbor Laboratory Press NATURE METHODS VOL.3 NO.1 JANUARY

5 20 Lyse the remaining cells by sonication until the cloudy suspension becomes translucent. 21 Collect the cell debris by centrifuging at 10,000g for 5 min and retain 10 µl of the supernatant for analysis by SDS-PAGE. Replace the remaining supernatant with an equivalent volume of TBS and resuspend the pellet by sonication. Retain 10 µl of the resuspended pellet for analysis by SDS-PAGE. 22 Analyze the preinduced sample, induced sample, supernatant and pellet of each colony using standard SDS-PAGE methods to determine expression levels and solubility (presence in the supernatant) either by Coomassie staining or western blotting. TROUBLESHOOTING 23 Once a suitable expression clone has been identified, use the glycerol stock to inoculate a starter culture in 100 ml LB supplemented with the appropriate antibiotics (depending on the plasmids present) and incubate at 37 C for h (or 30 C for longer than 15 h). Alternatively, inoculate the 100-ml culture with a colony from a fresh transformation of the expression clone (see Steps 12 and 13). 24 Use 1 5 ml of starter culture to inoculate 1 l of LB supplemented with the appropriate antibiotics in 2 l flasks. Multiple flasks can be used to scale-up expression. 25 Incubate at 37 C until A 600 is ~0.3. Then lower the temperature to between 18 and 30 C and continue to incubate until the A 600 is Remove 10 µl of the culture for analysis by SDS-PAGE (preinduced sample). 27 Induce the remaining culture by adding 1 ml of 1 M IPTG (final concentration can range from 0.1 to 1 mm) and continue to incubate for an additional 2 14 h. 28 Remove 10 µl of the culture for analysis by SDS-PAGE (induced sample). 29 Harvest the cells by centrifugation at 4,000g for 30 min and discard the supernatant. 30 Resuspend the cells in 10 ml of ice-cold TBS (supplemented with complete protease inhibitor cocktail and 1 mg/ml lysozyme) per liter of culture. Growth of cultures for large-scale expression Harvesting and lysis of the cells 31 Freeze at 70 C. PAUSE POINT At this point cells can be stored indefinitely. 32 Thaw the cells and lyse them by sonication until the cloudy suspension becomes translucent. 33 Collect the cell debris by centrifuging at 10,000g for 30 min and retain 10 µl of the supernatant for analysis by SDS-PAGE. Replace the remaining supernatant with an equivalent volume of TBS and resuspend the pellet by sonication. Retain 10 µl of the resuspended pellet for analysis by SDS-PAGE. Analysis of the lysate 34 Analyze the preinduced sample, induced sample, supernatant and pellet using standard SDS-PAGE methods to determine expression levels and solubility (presence in the supernatant). TROUBLESHOOTING Further purification of soluble targets can be achieved by affinity chromatography depending on the tags present. For additional information on affinity chromatography, see Molecular Cloning, Chapter 15, Protocols 5 7 ( Cold Spring Harbor Laboratory Press NATURE METHODS VOL.3 NO.1 JANUARY

6 TROUBLESHOOTING TABLE PROBLEM Step 13 Few or no transformants obtained. Steps 22 and 34 Low yield or partial solubility observed in both lysis pellet and supernatant. Target is completely insoluble (observed only in pellet). SOLUTION Transformation with multiple plasmids is sensitive to both the post-transformation recovery period and the concentration of antibiotic used for selection. For these experiements, make sure that transformants are allowed to recover for at least 1 h before plating and that the concentration of antibiotic is reduced to half of that recommended in Table 1. The yield and solubility of proteins are affected by several factors including the expression strain, IPTG concentration and the duration and temperature of induction. These factors should be systematically varied to optimize results. Expression of toxic proteins at a higher cell density (A 600 ) for a shorter period can help to increase yields. Attempt to add an additional interacting partner of the target to the coexpression system to aid in folding in an attempt to enhance solubility. Test various chaperones by coexpression for their ability to fold the target. Rapidly lowering temperature before induction can induce cold shock proteins and may result in target insolubility. Maintain a constant (but low) temperature during initial growth and induction. Modify the target by truncating and/or deleting sections of the coding sequence to obtain a domain that is soluble. If possible, switch to a homolog of the target from a different species that may be soluble. CRITICAL STEPS Step 4 The choice of vector is determined by whether or not the target is to be tagged. Usually only the primary target is tagged and ancillary factors are not tagged. An exception to this is a doubletagging method for double-affinity chromatography. In this case, two proteins of a complex are tagged with different tags. See Box 1 and Table 1 for various strategies and vector selection. The vectors described in this protocol contain several tags: Duet MCS1 contains an amino-terminal His tag, Duet MCS2 contains a carboxy-terminal S tag, pgex encodes an N-terminal GST tag and pmal encodes an N-terminal MBP tag. A major advantage of His, GST and MBP tags is that they allow affinity purification of the fusion protein with mild elution conditions. The pgex and pmal vectors also include various protease cleavage sites between the tag and the protein that can be used to remove the tag after purification. Although the S tag allows affinity purification, it requires harsher elution conditions and is not recommended for the purification of complexes under nondenaturing conditions. This protocol therefore focuses on His-, GST- and MBP-tag systems. The choices of which tag to use and which proteins to tag will influence the solubility and functionality of the product. Choices will be dictated by the experimental aims. His tags are small and have a low metabolic burden, but they can sometimes inhibit solubility 4. GST tags have no effect on solubility and MBP tags tend to increase the solubility of the target 5, but the large size of both these tags is metabolically expensive and may affect the function of the fusion protein. (The latter can be avoided if the tag is removed by proteolysis with a specific protease.) Lastly, GST forms dimers in solution, a feature that can be problematic, especially if the target protein also forms oligomers. The most advantageous protein to be tagged must be determined empirically, but tagging two proteins with the same tag is not recommended. This could result in heterogeneity of the purified proteins or complexes. Step 9 DH5α cells are used at this point for plasmid maintenance. DH5α is highly transformable and is reca ; that is, the cells have a low recombination rate allowing for the stable maintenance of plasmid DNA. 60 VOL.3 NO.1 JANUARY 2006 NATURE METHODS

7 Step 12 BL21 (DE3) is required at this point for several reasons. The Duet vectors have a T7 promoter for high-level expression and therefore require an E. coli host containing a chromosomal copy of the T7 RNA polymerase gene (DE3). pgex and pmal vectors both contain a tac promoter for high-level expression. Although the tac promoter does not require a DE3 strain (as they are recognized by E. coli RNA polymerase), coexpression of pgex or pmal with Duet vectors will necessitate the use of a DE3 strain. DE3 strains will not hinder expression from vectors containing the tac promoter. BL21 (DE3) strains are also preferred at this stage as they are deficient in OmpT and Lon proteases that could otherwise induce proteolysis of overexpressed proteins. Step 13 Transformation of cells with multiple plasmids may result in the maintenance of fewer copies of each plasmid, as compared to those seen in single-plasmid transformation. Reducing the concentration of antibiotics by half allows the survival of these low-copy-number transformants. Steps 17 and 25 Induction temperature can also influence target solubility. Lower temperatures favor slower expression rates that might allow for improved folding of the target, but will also reduce target yield. Different temperatures should be tested to determine the optimal temperature. Expression of toxic proteins can also lead to poor yields. Inducing expression of toxic proteins at a higher A 600 for a shorter period can increase yields. Steps 17 and 27 IPTG concentration and length of induction can both influence target solubility. Lower concentrations of IPTG may induce protein expression at a slower rate, allowing better folding. Lower IPTG concentrations, however, may also reduce yield. Longer induction times, which increase expression, may also induce proteolysis of the target, leading to heterogeneity in the sample and reducing yield. Different concentrations of IPTG and a time course of induction should be tested to determine the optimal combination. Step 23 Growth of starter cultures at 37 C for extended periods is not recommended because this can sometimes lead to plasmid silencing that prevents expression. COMMENTS Successful coexpression and purification of heterologous proteins is dependent on several factors, including plasmid compatibility, identity of the tag used (critical step 4), which protein of a complex is tagged, E. coli host strain and, of course, the nature of proteins being expressed. Several different strategies are possible (various strategies for coexpression are described in Box 1) depending on the requirements of the protein(s) to be expressed. Maintenance of multiple plasmids in E. coli is dependent on the plasmids possessing compatible origins of replication 6 and different antibiotic resistance genes for selection (Table 1). If two plasmids containing the same origin of replication are used to cotransform E. coli, the replication of one plasmid will be inhibited as a result of an RNA antisense mechanism, which causes plasmid segregation within the population 6,7. Compatible plasmids stably coexist in a single cell by occupying different subcellular locations within the bacterium 8. Plasmid segregation can also occur if the plasmids carry the same antibiotic resistance gene; only one plasmid needs to be maintained to confer resistance. Therefore, plasmids sharing common origins of replication or antibiotic resistance genes should not be used for the coexpression of two proteins. Because the pgex and pmal vectors are ColE1-based and ampicillin-resistant, they are incompatible with petduet. Thus, if the primary target is to be tagged with either GST or MBP, the plasmids prsfduet, pcoladuet, pacycduet or pcdfduet should be used for the expression of additional targets. See Table 1 for information on plasmid compatibility. EXAMPLE OF APPLICATION Recent studies have linked Argonaute (Ago) proteins as the catalytic core of the RNA-induced silencing complex (RISC), the RNA interference (RNAi) effector complex. The crystal structure of Pyrococcus furiosus Argonaute revealed that the signature PIWI domain of the Ago proteins is similar to that of ribonuclease (RNase) H and contains the conserved catalytic residues 9. Purification of tagged Ago from human cell lines revealed that endonuclease (or slicer ) activity was associated with complexes containing Ago2 (refs. 10,11), and that mutation of the conserved catalytic residues abolishes activity 10. Cold Spring Harbor Laboratory Press NATURE METHODS VOL.3 NO.1 JANUARY

8 BOX 1 STRATEGIES FOR COEXPRESSION The Duet/pGEX/pMAL system described in this protocol offers several optimal options, tailored to the needs of various targets (Table 2). One protein plus refolding factor. This approach involves the coexpression of a tagged protein with a chaperone, or cofactor, to improve folding and/or solubility of the product (see the Example of application section and Fig. 2). Several different chaperones should be tested to find the optimal partner for the protein of interest. The first strategy (Table 2; target in Duet, factor in Duet) has the advantage that the primary target and the refolding factor can be carried on a single plasmid. Having the two proteins on the same plasmid makes the testing of protein variants (for example, truncations or deletions) cumbersome, because each variant of each protein must be generated and then recloned into the plasmid. This problem can be relieved by using two compatible plasmids, which would allow variants to be tested by mixing and matching plasmids, but this would introduce the complication of generating multiple transformants. The other two strategies (Table 2; target in pgex or pmal, factor in Duet) are similar, but use two plasmids. They offer the same advantages of tagging (here the GST or MBP tags are used to ease purification and improve product solubility), together with easy testing of protein variants, but require multiple transformations, which can be difficult. Single-tagged two-protein complex. A complex of two proteins can be expressed (on one or two plasmids) and purified using an affinity tag on only one of the proteins. The strategies are essentially as described above for the one protein plus refolding factor example. Double-tagged two-protein complex. Double tagging is a convenient two-plasmid method for the purification of a two-protein complex. It involves using different tags on each of the proteins, and sequential affinity chromatography to purify the complex. Double tagging also allows the separate recovery of individual proteins, if desired. With the vectors described here, only GST tag His tag or MBP tag His tag combinations are recommended because of limitations of plasmid compatibility. Other tags can be used as long as plasmid compatibility is maintained. These strategies have the same advantages and disadvantages described for the one protein plus refolding factor example using two plasmids. Two-protein complex plus refolding factor. This approach is analogous to the two-protein complex discussed above, with the addition of an ancillary factor to aid the solubility and/or folding of one of the proteins. Because the ancillary factor will not be recovered, it can be left untagged. Tagging the two proteins to be recovered will assist in their purification and could also improve their solubility. Because each plasmid may have a different subcellular localization within the bacterium 6, cloning the refolding factor and its target protein on the same Duet vector may prove more effective as the two proteins then should localize within the same region of the cell. Multiprotein complex. A multiprotein complex can be built up from a two-protein complex by introducing additional proteins on other Duet vectors. Care should be taken when choosing a tagging strategy; multiple His tags used (in MCS1) may cause heterogeneity of the purified complexes and should be avoided. The Duet system allows up to eight proteins to be coexpressed simultaneously, but this number is reduced to only seven if either the pgex or pmal vectors are used. Figure 2 Example of application. Coexpression of pgex-4t-1 harboring the gene encoding GST-hAgo2 with prsf-duet harboring the gene encoding hhsp90 results in soluble expression of GST-hAgo2 (right, E; red arrow). Analysis by SDS-PAGE shows that expression of pgex-4t-1-hago2 alone yields insoluble GST-Ago2 (left lanes WCL, P; asterisk) and no soluble GSTtagged hago2 (left, E). Soluble hhsp90 can be observed in the supernatant (right, S; black arrow). The presence of soluble GST-hAgo2 was verified by mass spectrometry and was shown to be active 2. WCL, whole-cell lysate; P, pellet; S, supernatant; E, eluate from the glutathione affinity step. 62 VOL.3 NO.1 JANUARY 2006 NATURE METHODS

9 To definitively prove that Ago2 itself is the slicer enzyme and to further characterize its activity, we sought to obtain purified human Ago2 from E. coli for biochemical characterization because E. coli does not possess an RNAi pathway. However, expression of GST-tagged human Ago2 (GST-hAgo2) in E. coli yielded insoluble protein (Fig. 2). Ago2-containing complexes purified from human cell lines contained almost stoichiometric amounts of human Ago2 and human heat shock protein 90 (hhsp90; ref. 10). We therefore tested whether coexpression of GST-hAgo2 with hhsp90 in E. coli could overcome the insolubility resulting in functional protein. Indeed, we obtained soluble GST-hAgo2 using this method (Fig. 2) 2. With this system, we demonstrated that GST-hAgo2 alone can combine with a small interfering RNA (sirna) to form a recombinant complex (termed minimal RISC) that can accurately cleave substrate RNAs and that this complex has similar kinetic properties to both fruit fly and human RISC 2. A major difference between these complexes, however, is that, unlike fly or human RISC, minimal RISC is not stimulated by ATP, indicating that factors stimulating product release are absent from the recombinant complex. These studies demonstrate that Ago2 catalyzes mrna cleavage within RISC, and pave the way for complete recombinant reconstitution of the RNAi effector complex. Table 2 Strategies for coexpressing different targets One protein plus refolding factor Target protein Factor Vector (MCS) Tag Vector (MCS) Tag Notes Duet (MCS1) His Duet (MCS2) None Both MCSs can be used on a single Duet vector, or two compatible vectors could be used. pgex GST Duet (MCS2) None Any Duet vector can be used, except pet-duet because of incompatibility with pgex. pmal MBP Duet (MCS2) None Any Duet vector can be used, except pet-duet because of incompatibility with pmal. Single-tagged two-protein complex Protein 1 Protein 2 Vector (MCS) Tag Vector (MCS) Tag Notes Duet (MCS1) His Duet (MCS2) None Both MCSs can be used on a single Duet vector, or two compatible vectors could be used. pgex GST Duet (MCS2) None Any Duet vector can be used, except pet-duet because of incompatibility with pgex. pmal MBP Duet (MCS2) None Any Duet vector can be used, except pet-duet because of incompatibility with pmal. Double-tagged two-protein complex Protein 1 Protein 2 Vector (MCS) Tag Vector (MCS) Tag Notes pgex GST Duet (MCS1) His Any Duet vector can be used, except pet-duet because of incompatibility with pgex. pmal MBP Duet (MCS1) His Any Duet vector can be used, except pet-duet because of incompatibility with pmal. Two-protein complex plus refolding factor Protein 1 Protein 2 Factor Vector (MCS) Tag Vector (MCS) Tag Vector (MCS) Tag Notes pgex GST Duet (MCS1) His Duet (MCS2) None Any Duet vector can be used, except pet-duet because of incompatibility with pgex. Both MCSs can be used on a single Duet vector, or two compatible vectors could be used. pmal MBP Duet (MCS1) His Duet (MCS2) None Any Duet vector can be used, except pet-duet because of incompatibility with pmal. Both MCSs can be used on a single Duet vector, or two compatible vectors could be used. Cold Spring Harbor Laboratory Press NATURE METHODS VOL.3 NO.1 JANUARY

StrataPrep Plasmid Miniprep Kit INSTRUCTION MANUAL Catalog #400761 and #400763 Revision A For In Vitro Use Only 400761-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this

Purification and Characterization of a DNA Plasmid Part A CHEM 4581: Biochemistry Laboratory I Version: January 18, 2008 INTRODUCTION DNA Plasmids. A plasmid is a small double-stranded, circular DNA molecule

Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab With the Gateway cloning system, a PCR fragment is

Diagnosis Sanger Interpreting and Troubleshooting Chromatograms GENEWIZ Technical Support DNAseq@genewiz.com Troubleshooting This troubleshooting guide is based on common issues seen from samples within

ChIP is a powerful tool that allows the specific matching of proteins or histone modifications to regions of the genome. Chromatin is isolated and antibodies to the antigen of interest are used to determine

Product Manual Rapid GST Inclusion Body Solubilization and Renaturation Kit Catalog Number AKR-110 FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Bacteria are widely used for His

Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning This experiment was designed by Dylan Dodd, based on research completed in Dr. Isaac Cann s lab*, with modifications and editing of content

Cat. Nos. C400EL10, C400CH10, and CIS40025 CopyCutter EPI400 Electrocompetent and Chemically Competent E. coli* cells were developed to significantly lower the copy number of a wide variety of common vectors

CHAPTER 9 DNA Technologies Recombinant DNA Artificially created DNA that combines sequences that do not occur together in the nature Basis of much of the modern molecular biology Molecular cloning of genes

for preparation of 100 nucleic acid samples Cat. No. 1 796 88 Principle Cells are lysed during a short incubation with Proteinase K in the presence of a chaotropic salt (guanidine HCl), which immediately

Product Manual Rapid GST Inclusion Body Solubilization and Renaturation Kit Catalog Number AKR-110 FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Bacteria are widely used for His

GST Purfication and Pulldown Part I Instructor: David Deitcher TA: Kristy Lawton In order to study the function of a protein it is often useful to have that protein purified away from others in the cell.

Lambda CE6 Induction Kit INSTRUCTION MANUAL Catalog #235200 Revision A For In Vitro Use Only 235200-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product. No other

1. Purpose 1.1. The purpose of this protocol is to transfer a transgene from the pshuttlex plasmid to padenox. 1.2. The starting material is 10 μg plasmid DNA. 1.3. This procedure is routinely performed

Genetics and Genomics in Medicine Chapter 3 Multiple Choice Questions Questions & Answers Question 3.1 Which of the following statements, if any, is false? a) Amplifying DNA means making many identical

Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single

Construction of pcxz14w, a Novel puc19-derived Plasmid Encoding the rop Gene Shary Chen, Ziyan Xu, Wenchen Zhao Department of Microbiology and Immunology, University of British Columbia When the ColE1-derived

HiPer Transformation Teaching Kit Product Code: HTBM017 Number of experiments that can be performed: 10 Duration of Experiment: 4 days Day 1- Preparation of media and revival of E. coli Host Day 2- Inoculation

Laboratory for Environmental Pathogens Research Department of Environmental Sciences University of Toledo Polymerase Chain Reaction (PCR) Background information The polymerase chain reaction (PCR) is an

Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive to ionizing radiation. However, why these mutants are

QIAfilter Plasmid Midi Kit (Cat #: 12243) Things to do before starting Add the provided RNase A solution to Buffer P1 before use. Use one vial of RNase A (centrifuge briefly before use) per bottle of Buffer

The Production of a Recombinant Biotechnology Product Chapter 8 Objectives Give a basic overview of genetic engineering. Describe the processes involved in isolating a piece DNA of interest Mass producing

Name KEY Section Biology 201 (Genetics) Exam #3 120 points 20 November 2006 Read the question carefully before answering. Think before you write. You will have up to 50 minutes to take this exam. After